Insights into the bacterial symbiont diversity in spiders

Abstract Most spiders are natural enemies of pests, and it is beneficial for the biological control of pests to learn the relationships between symbionts and their spider hosts. Research on the bacterial communities of insects has been conducted recently, but only a few studies have addressed the bacterial communities of spiders. To obtain a complete overview of the microbial communities of spiders, we examined eight species of spider (Pirata subpiraticus, Agelena difficilis, Artema atlanta, Nurscia albofasciata, Agelena labyrinthica, Ummeliata insecticeps, Dictis striatipes, and Hylyphantes graminicola) with high‐throughput sequencing based on the V3 and V4 regions of the 16S rRNA gene. The bacterial communities of the spider samples were dominated by five types of endosymbionts, Wolbachia, Cardinium, Rickettsia, Spiroplasma, and Rickettsiella. The dominant OTUs (operational taxonomic units) from each of the five endosymbionts were analyzed, and the results showed that different spider species were usually dominated by special OTUs. In addition to endosymbionts, Pseudomonas, Sphingomonas, Acinetobacter, Novosphingobium, Aquabacterium, Methylobacterium, Brevundimonas, Rhizobium, Bradyrhizobium, Citrobacter, Arthrobacter, Pseudonocardia, Microbacterium, Lactobacillus, and Lactococcus were detected in spider samples in our study. Moreover, the abundance of Sphingomonas, Methylobacterium, Brevundimonas, and Rhizobium in the spider D. striatipes was significantly higher (p < .05) than the bacterial abundance of these species in seven other spider species. These findings suggest that same as in insects, co‐infection of multiple types of endosymbionts is common in the hosts of the Araneae order, and other bacterial taxa also exist in spiders besides the endosymbionts.

. Spiders are perceived as important natural enemies for pests (Marc, Canard, & Ysnel, 1999;Nyffeler & Sunderland, 2003), and many researchers have focused their attention on the endosymbionts infection of spiders (Duron et al., 2008;Goodacre et al., 2006;Rowley, Raven, & McGraw, 2004) and the relationships between the endosymbionts (such as Wolbachia, Cardinium, Rickettsia and Spiroplasma) and their spider hosts (Curry, 2013;Gunnarsson, Goodacre, & Hewitt, 2009;Martin & Goodacre, 2009). In regard to the bacterial community of spiders, only a few studies have been reported (Vanthournout & Hendrickx, 2015;Zhang, Zhang, Yun, & Peng, 2017). Vanthournout  Comparing the prevailing research on the bacterial community of insects, the bacterial communities of only single spider species have been examined. To provide insights into the bacterial diversity of multiple spiders (especially the bacteria not belonging to endosymbionts), in this study, we detected the bacterial diversity of eight spider species using a high-throughput sequencing technique, and through the distribution and relative abundance of different bacteria in different spider species, we analyzed the difference in bacterial communities among all eight spider species. By revealing the other bacteria (besides endosymbionts) in spiders, this research on the symbionts of spiders will add to the understanding of all bacteria (such as gut bacteria or environmental bacteria) besides common endosymbionts.

| Sample collection
In the summer of 2016, three species of spiders (Pirata subpiraticus, Agelena difficilis, and Artema Atlanta) were collected near Shahu, Wuhan (China), and three species of spiders (Nurscia albofasciata, Agelena labyrinthica, and Ummeliata insecticeps) were collected near Shizishan, Wuhan (China). Dictis striatipes was collected in Guangpo, Lingshui (China), and Hylyphantes graminicola was collected in Longmen, Luoyang (China; See Table 1). Ten individuals were collected for each spider species, and all spiders collected in this study were ecologically important species (Zhang & Wang, 2017). The species were identified based on the morphological features of the specimens. Living samples were transported to the laboratory and starved for 2 weeks. Then, samples were fixed in 100% ethanol and stored at −20°C. All eight spider species used in this study were identified as a nonendangered and nonprotected species.

| DNA extraction
Each sample was cleaned using an ultrasonic cleaner (FRQ-1004T) filled with a 75% alcoholic solution for 1-2 min to remove surface bacteria and pollutants, followed by three washes with sterile ultrapure water. The DNA was extracted from each individual (whole body) using the QIAGEN DNeasy Kit (Germany) following the manufacturer's recommended protocol. DNA was then quantified using a nanophotometer (NanoPhotometer NP80 Touch, Implen GmbH). An equimolar amount of DNA from each of the two individuals of the same species was mixed into one of the DNA pools. The name of the DNA groups and the numbers of DNA pools in each spider species are shown in Table 1. TA B L E 1 Spider samples used in this study and elongation at 72°C for 45 s. For the last cycle, the elongation time was extended to 7 min at 72°C. PCR products were run on 2% agarose gels, and the samples producing visualized amplicons were utilized for high-throughput sequencing of microbial diversity. The variable region V3-V4 of the 16S rDNA was used to assess bacterial diversity (Caporaso et al., 2012). The sequencing was conducted on an Illumina HiSeq platform at BioMarKer Technologies Co. Ltd.

| Bioinformatic analyses
Paired-end reads were merged into single, longer sequences using FLASH version 1.2.7 (Magoč & Salzberg, 2011). Quality filtering on the raw tags was performed under specific filtering conditions (The Sliding Window uses 50 bp. This works by scanning from 5′ end of the read and removes the 3′ end of the read when average quality of a group of bases drops below 20 bp to obtain high-quality clean tags by Trimmomatic version 0.33 (Bolger, Lohse, & Usadel, 2014). UCHIME version 4.2 (default setting: 80% similarity) was used to identify and eliminate chimeric sequences (Edgar, Haas, Clemente, Quince, & Knight, 2011). The remaining sequences were assigned into operational taxonomic units (OTUs) at 97% similarity using UCLUST version 1.2.22 (Edgar, 2010). The taxonomic identification of each OTU was conducted by comparing the representative sequences (the sequences which has the most highest relative abundance) of each cluster against SILVA by a BLASTn search (Quast et al., 2013), and the taxonomic classification of each OTU was performed using Ribosomal Database Project (RDP) Classifier version 2.2 with the classification threshold set at 0.8 (Cole et al., 2009

| Statistical analyses
Differences in the relative abundance of certain bacterial types among different groups were analyzed with the Mann-Whitney U test. All of the data showed that the microbiota of the present spider species had a high diversity (Figure 1). The dissimilarity between the bacterial communities of samples was quantified by the Bray-Curtis distance. Principal coordinate analysis (PCoA) showed that the bacterial communities were much more similar within species than between species ( Figure 2).  Table 2).

| The dominant endosymbiont OTUs in spiders
Different OTU types of endosymbionts prevailed in different spider hosts (See Figure 3,

| The other bacterial taxa of bacterial communities in spider hosts
In addition to endosymbionts, there were other bacteria in the bacterial communities of spiders. Data were shown as the mean ± SE. The data were compared using a nonparametric Kruskal-Wallis test, which tests for differences between different groups. The relative abundance of each bacterial taxa was tested using the significant difference between groups when p < .05. D, K, M, O, P, R, S, and T indicate spider species P. subpiraticus, N. albofasciata, D. striatipes, A. labyrinthica, A. difficilis, A. atlanta, U. insecticeps, and H. graminicola, respectively. The shaded and bold values indicate that the relative abundance of Sphingomonas, Methylobacterium, Brevundimonas, and Rhizobium in spider D. striatipes was significantly higher (p < .05) than the bacterial abundance in seven other kinds of spiders Pseudonocardia, and Microbacterium, were found in all of the samples in our study. Lactobacillus and Lactococcus, which belong to the phylum Firmicutes, were detected in spiders except D. striatipes and H. graminicola (Table 2). Moreover, the abundance of Sphingomonas, Methylobacterium, Brevundimonas, and Rhizobium in spider D. striatipes was significantly higher (p < .05) than the bacterial abundance in seven other kinds of spiders, and no differences were obtained for these four bacteria between the other seven spider species (Table   S2).

| D ISCUSS I ON
The bacterial community of a single kind of spider has been previ- Co-infection of multiple endosymbionts in the arthropods host was common (Duron et al., 2008;Engelstädter & Hurst, 2009;Goodacre et al., 2006), and relatively few studies have explored the phenotypic effect of multiple endosymbionts on their hosts (Curry, Paliulis, Welch, Harwood, & White, 2015;White, Kelly, Cockburn, Perlman, & Hunter, 2011 Spiders have a special feeding mode. They usually bite part of the prey and then quickly inject venom into the body of prey and sucked the prey (Foelix, 2011). We hypothesize that the gut bacteria of spiders may be distinct from insects or other species in Arachnoidea (such as mites and scorpions). However, there have been no reports regarding the gut bacteria communities of spiders until now. Almost all of the nonendosymbionts present (except Pseudonocardia) in our study were detected in the gut of some insects (Anjum et al., 2017;Gupta et al., 2014;Snyman, Gupta, Bezuidenhout, Claassens, & van den Berg, 2016;Wang, Gilbreath, Kukutla, Yan, & Xu, 2011). Moreover, the bacteria from genus Pseudomonas, Citrobacter and Lactococcus were also found in the gut of a predatory beetle Poecilus chalcites (Lehman, Lundgren, & Petzke, 2009) Also as a kind of predator, the gut bacteria of spiders may be similar with the gut bacterial structure of predatory insects. From our results, we suppose that the gut bacteria of spiders may be composed by indigenous bacteria and environmental bacteria, and the relative abundance of bacteria within their hosts is related to the hosts' species and the environment of the hosts.

ACK N OWLED G M ENTS
We thank Fengxiang Liu for help with the sample collection. This work was supported by the National Natural Science Fund of China (31401982, 31672317) and the Key Scientific and Technological Projects of Hubei (2016AHB003).

CO N FLI C T O F I NTE R E S T
All of the authors declare that they have no conflict of interest in the publication.

AUTH O R CO NTR I B UTI O N S
YUELI YUN and YU PENG designed the experiments. LIHUA ZHANG and GUOWEN HU conducted the experiments and data analysis.
YUELI YUN and LIHUA ZHANG wrote the manuscript.

DATA ACCE SS I B I LIT Y
The original data of the symbionts relative abundance in spiders are available from the Dryad Digital Repository: https://doi. org/10.5061/dryad.7k702.